Human cytomegalovirus (HCMV) is the leading cause of viral congenital infections worldwide, involving about 1% of newborns. In children, the consequences may be severe, especially in case of maternal primary infection during pregnancy. In the United States, about 30,000 to 40,000 newborns are affected each year; more than 9,000 of these children are left with permanent neurological sequelae. Demmler, G. J., 1994, Congenital cytomegalovirus infection, Semin. Pediatr. Neurol. 1(1):36-42. The annual cost of treating cytomegalovirus infection complications in the United States is about two billion dollars. Daniel Y. et al., 1995, Congenital cytomegalovirus infection, Eur. J. Obstet. Gynecol. Reprod. Biol., 63(1):7-16.
HCMV is a species-specific member of the herpes virus family. Other well-known members of the herpes virus family include herpes simplex virus, types I and II, Epstein-Barr virus and Varicella Zoster virus. Although these viruses are related to each other taxonomically as double-stranded DNA viruses, infections due to these viruses manifest in a clinically distinct manner. In the case of HCMV, medical conditions arising from congenital infection include jaundice, respiratory distress, and convulsive seizures which may result in mental retardation, neurologic disability or death. As noted above, congenital HCMV infection produces significant problems from both personal and public health perspectives.
Infection in adults is frequently asymptomatic, but may manifest as mononucleosis, hepatitis, pneumonitis, or retinitis. HCMV infection is particularly significant in immunocompromised patients such as AIDS sufferers, chemotherapy patients, and organ transplant patients undergoing tissue rejection therapy.
The mechanisms of HCMV pathogenesis are not fully understood. It is believed that host factors, such as cellular and/or humoral immune responses might be involved. See, Alford and Britt, xe2x80x9cThe Human Herpesvirusesxe2x80x9d, Eds. Roizman, B., R. J. Whitley and C. Lopez, Raven Press, N.Y., 1993, pp 227-55. It has also been speculated that genetic variability (either structural or antigenic or both) among different strains of HCMV could be responsible for the variability in clinical manifestations observed. See, Pritchett, R. F., 1980, DNA nucleotide sequence heterogeneity between the Towne and AD169 strains of cytomegalovirus, J. Virol. 36(1):152-61; Lehner, R. et al., 1991, Comparative sequence analysis of human cytomegalovirus strains, J. Clin. Microbiol. 29(11):2494-502; Fries, B. C., 1994, Frequency distribution of cytomegalovirus envelope glycoprotein genotypes in bone marrow transplant recipients, J. Infect. Dis. 169(4):769-74.
Classical drug therapies have generally focused upon interactions with proteins in efforts to modulate their disease causing or disease potentiating functions. Such therapeutic approaches have failed for cytomegalovirus infections.
Effective therapy for HCMV has not yet been developed despite studies on a number of antiviral agents. Interferon, transfer factor, adenine arabinoside (Ara-A), acycloguanosine (Acyclovir, ACV), and certain combinations of these drugs have been ineffective in controlling HCMV infections. Based on preclinical and clinical data, foscarnet (PFA) and ganciclovir (DHPG) show limited potential as antiviral agents. PFA treatment has resulted in the resolution of HCMV retinitis in five AIDS patients to date. DHPG studies have shown efficacy against HCMV retinitis and colitis. DHPG seems to be well tolerated by most treated individuals, but the ppearance of a reversible neutropenia, the emergence of resistant strains of HCMV upon long-term administration, and the lack of efficacy against HCMV pneumonitis limit the long term applications of this compound.
Immunoglobulin has also been utilized for treating HCMV infections. See, Condie, R. M. et al., 1984, Prevention of cytomegalovirus infection in bone marrow transplant recipients by prophylaxis with an intravenous, hyperimmune cytomegalovirus globulin, Birth Defects, 20:327-344; Perrillo, R. P. et al., 1987, Immune globulin and hepatitis B immune globulin, Arch. Intern Med., 144:81-85; Snydman, D. R., et al. 1987, Use of cytomegalovirus immune globulin to prevent cytomegalovirus disease in renal-transplant recipients, N. Engl. J. Med., 317:1049-1054. The development of more effective and less-toxic therapeutic compounds and methods is needed for both acute and chronic use.
Several HCMV vaccines have been developed or are in the process of development. Vaccines based on live attenuated strains of HCMV have been described. See, Plotkin, S. A. et al., 1984, Lancet, 1:528-30; Plotkin, S. A. et al., 1976, J. Infect. Dis., 134:470-75; Glazer, J. P. et al., 1979, Ann. Intern. Med., 91:676-83; and U.S. Pat. No. 3,959,466. A proposed HCMV vaccine using a recombinant vaccinia virus expressing HCMV glycoprotein B has also been described. Cranage, M. P. et al., 1968 EMBO J., 5:3057-3063. However, vaccinia models for vaccine delivery are believed to cause local reactions. Additionally, vaccinia vaccines are considered possible causes of encephalitis.
Adenoviruses have been developed previously as efficient heterologous gene expression vectors. For example, an adenovirus vector has been employed to express herpes simplex virus glycoprotein gB (Johnson, D. C. et al., 1988, Virol., 164:1-14), human immunodeficiency virus type 1 envelope protein (Dewar, R. L. et al., 1988, J. Virol., 63:129-136), and hepatitis B surface antigen (Davis, A. R. et al, 1985, Proc. Natl. Acad. Sci., U.S.A., 82:7560-7564; Morin, J. E. et al., 1987, Proc. Natl. Acad. Sci., U.S.A., 84:4626-4630). Adenoviruses have also been found to be non-toxic as vaccine components in humans (Takajuji, E. T. et al., 1970, J. Infect. Dis., 140:48-53; Collis, P. B. et al., 1973, J. Inf. Dis., 128:74-750; and Couch, R. B. et al., 1963, Am. Rev. Respir. Dis., 88:394-403). U.S. Pat. Nos. 5,591,439 and 5,552,143 provide novel vaccine components for HCMV which comprise an adenovirus expression system capable of expressing a selected HCMV subunit gene in vivo.
Human CMV is a large, enveloped herpesvirus whose genome consists of a double-stranded DNA molecule approximately 240,000 nucleotides in length. This genome is the most complex of all DNA viruses and is approximately 50% larger than the genome of herpes simplex virus (HSV). Intact viral DNA is composed of contiguous long (L) and short (S) segments, each of which contains regions of unique DNA sequence flanked by homologous regions of repetitive sequence. As a group, human CMV isolates share at least 80% sequence homology, making it nearly impossible to classify cytomegaloviruses into subgroups or subtypes, although variations in the restriction endonuclease patterns of various CMV DNA preparations are identifiable in epidemiologically unrelated strains. The DNA of the prototypic strain of CMV (AD 169) has been sequenced and reported to contain a conservative estimate of 175 unique translational open reading frames (ORFs). A number of the predicted CMV gene products show homology to other human herpesvirus gene products.
The large genome of CMV is difficult to manipulate. Cloning and mutagenesis of murine CMV (MCMV) has been accomplished using a bacterial artificial chromosome (xe2x80x9cBACxe2x80x9d). See, Messerle, et al. 1997, Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. U.S.A., 94: 14759-14763. Cloning of MCMV in a BAC allows for manipulation of the CMV genome within the bacterial system. Another useful vector, the yeast artificial chromosome (xe2x80x9cYACxe2x80x9d) has been utilized to clone an infectious adenovirus (Ketner et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:6180-6190). However, it has not yet been demonstrated that the CMV genome could be successfully cloned into and manipulated within a yeast artificial chromosome (xe2x80x9cYACxe2x80x9d).
Yeast artificial chromosomes (YACs) allow the propagation of very large segments of exogenous DNA in a microbial organism that is easy to work with and grow (Schlessinger, D. 1990, Yeast artificial chromosomes: tools for mapping and analysis of complex genomes, Trends In Genetics 6:248-253). The great promise that YAC technology holds for studies of gene function and regulation has been strengthened by the development of methods for transferring YACs into mammalian cells and animals (Choi, T. K. et al., 1993, Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome. Nature Gen. 4:117-123).
YAC transgenic approaches are very powerful and are greatly enhanced by the ability to efficiently manipulate the cloned DNA. A major technical advantage of yeast is the ease with which specific genome modifications can be made via DNA-mediated transformation and homologous recombination. In particular, an alternative recombinant cloning method of YAC construction has recently been described (Ramsay, M. 1994, Yeast artificial chromosome cloning. Mol. Biotech. 1:181-201. The method involves co-transformation of a target DNA, such as the HCMV viral genome, with YAC vector arms containing appropriate DNA segments homologous to the target DNA. Recombination in the yeast cell then generates the desired YAC. A schematic of this procedure is shown in FIG. 1.
The present invention provides a YAC vector that includes the full-length DNA sequence of the CMV virus. This vector provides a solution to the long-felt need for a CMV vaccine, and provides a tool for studying the replication of CMV using cell-line models.
The present invention provides a YAC vector that includes portions of and full-length DNA sequence of the CMV virus. This vector provides a solution to the long-felt need for a CMV vaccine, and provides a tool for studying the replication of CMV using cell-line models.
The present invention provides recombinant DNA constructs and YAC vectors comprising, in certain embodiments, overlapping segments of the HCMV genome. In other embodiments, the present invention provides a recombinant DNA constructs and YAC vectors comprising the entire CMV genome. This invention provides stable partial and full length YAC-HCMV clones useful for studying CMV replication, generating CMV virus and constructing CMV vaccines.
In general, the present invention provides an isolated recombinant DNA molecule comprising a yeast artificial chromosome, including at least a portion of HCMV genome. In preferred embodiments the DNA molecule further comprises at least one a sequence. Preferably the HCMV genome is derived from the Towne strain or from the AD169 strain. In preferred embodiments, the recombinant DNA molecule comprises a DNA molecule selected from the group consisting of Y1-2, Y3-2, Y2-4, Y4-7 and Y5-26, described in Table 2, below. In another embodiment, the invention provides a composition suitable for use as a vaccine comprising the isolated recombinant DNA molecule and a pharmaceutically acceptable excipient.